Optimisation of enzymatic saccharification of wheat straw pre-treated with sodium hydroxide

To enhance the reducing sugar yield in enzymatic hydrolysis, various factors (NaOH concentration, solid content and pre-treatment time) that affect the pre-treatment process were investigated and evaluated based on the reducing sugar yield of the subsequent enzymatic hydrolysis. The enzymatic hydrolysis was based on the cellulase from Trichoderma reesi ATCC 26921, the optimum NaOH pre-treatment conditions were an NaOH concentration of 1.0% (w/w), a solid content of 5.0% (w/v) and a pre-treatment time of 60 min. Various parameters that affect the enzymatic hydrolysis of wheat straw, including the solid content, enzyme loading, pH and hydrolysis time, were investigated and optimized through a Box–Behnken design and response surface methodology. The predicted optimum conditions for enzymatic hydrolysis were a solid content of 8.0% (w/v), an enzyme loading of 35 FPU/g substrate, a temperature of 50 °C, a pH of 5.3 and a hydrolysis time of 96 h. The experimental result showed that the maximum reducing sugar yield was 60.73% (53.35% higher than the wheat straw without NaOH pre-treatment), which is in accordance with the predicted conditions.

Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading

Enzyme-linked immunosorbent assay (ELISA) is widely used in the routine screening of mycotoxin contamination in various agricultural and food products. Herein, a cascade-amplifying system was introduced to dramatically promote the sensitivity of an immunoassay for ochratoxin A (OTA) detection. Specifically, a biotinylated M13 bacteriophage was introduced as a biofunctional competing antigen, in which a seven-peptide OTA mimotope fused on the p3 protein of M13 was used to specifically recognize an anti-OTA monoclonal antibody, and the biotin molecules modified on capsid p8 proteins were used in loading numerous streptavidin-labeled polymeric horseradish peroxidases (HRPs). Owing to the abundance of biotinylated p8 proteins in M13 and the high molar ratio between HRP and streptavidin in streptavidin-polyHRP, the loading amount of HRP enzymes on the M13 bacteriophage were greatly boosted. Hence, the proposed method exhibited high sensitivity, with a limit of detection of 2.0 pg/mL for OTA detection, which was 250-fold lower than that of conventional ELISA. In addition, the proposed method showed a slight cross-reaction of 2.3% to OTB, a negligible cross-reaction for other common mycotoxins, and an acceptable accuracy for OTA quantitative detection in real corn samples. The practicability of the method was further confirmed with a traditional HRP-based ELISA method. In conclusion, the biotinylated bacteriophage and polyHRP structure showed potential as a cascade-amplifying enzyme loading system for ultra-trace OTA detemination, and its application can be extended to the detection of other analytes by altering specific mimic peptide sequences.

Strategies for the Immobilization of Eversa® Transform 2.0 Lipase and Application for Phospholipid Synthesis

Eversa® Transform 2.0 lipase (ET2) is a recent lipase formulation derived from the Thermomyces lanuginosus lipase cultivated on Aspergillus oryzae and specially designed for biodiesel production. Since it has not been available for a long time, research on the efficiency of this enzyme in other applications remains unexplored. Moreover, even though it has been launched as a free enzyme, its immobilization may extend the scope of ET2 applications. This work explored ET2 immobilization on octadecyl methacrylate beads (IB-ADS-3) and proved the efficiency of the derivatives for esterification of glycerophosphocholine (GPC) with oleic acid in anhydrous systems. ET2 immobilized via interfacial activation on commercial hydrophobic support Immobead IB-ADS-3 showed maximum enzyme loading of 160 mg/g (enzyme/support) and great stability for GPC esterification under 30% butanone and solvent-free systems. For reusability, yields above 63% were achieved after six reaction cycles for GPC esterification. Considering the very high enzyme loading and the number of reuses achieved, these results suggest a potential application of this immobilized biocatalyst for esterification reactions in anhydrous media. This study is expected to encourage the exploration of other approaches for this enzyme, thereby opening up several new possibilities.

Combining genetically engineered oxidase with hydrogen bonded organic framework (HOF) for highly efficient biocomposites.

Hydrogen bonded organic frameworks (HOFs) with enzymes incorporated during their bottom-up synthesis represent functional biocomposites with promising applications in catalysis and sensing. High enzyme loading while preserving high specific activity is fundamental for development, but to combine these biospecific features with a porous carrier is an unmet challenge. Here, we explored synthetic incorporation of D-amino acid oxidase (DAAO) with metal-free tetraamidine/tetracarboxylate-based BioHOF-1. Comparison of different DAAO forms in BioHOF-1 incorporation revealed that N-terminal enzyme fusion with the positively charged module Zbasic2 (Z-DAAO) promotes the loading (2.5-fold; ~500 mg g-1) and strongly boosts the activity (6.5-fold). To benchmark the HOF composite with metal-organic framework (MOF) composites, Z-DAAO was immobilized into the zeolitic imidazolate framework-8 (ZIF-8), the relatively more hydrophilic analogue metal azolate framework-7 (MAF-7). While sensitivity to the framework environment limited the activity of DAAO@MAF-7 (3.2 U mg-1) and DAAO@ZIF-8 (≤ 0.5 U mg-1), the activity of DAAO@BioHOF-1 was comparable (~45%) to that of soluble DAAO (50.1 U mg-1) and independent of the enzyme loading (100 – 500 mg g-1). The DAAO@BioHOF-1 composites showed superior activity with respect to every reported carrier for the same enzyme and excellent stability during solid catalyst recycling. Collectively, our results show that the fusion of the enzyme with a positively charged protein module enables the synthesis of highly active HOF biocomposites suggesting the use of genetic engineering for the preparation of biohybrid systems with unprecedented properties.

IMMOBILIZATION OF CELLULASES ON CHITOSAN: APPLICATION FOR SUGARCANE BAGASSE HYDROLYSIS

In this work, a commercial cellulolytic cocktail was immobilized on glutaraldehyde activated chitosan gel. The chitosan concentration in the gel preparation, pH, immobilization time and enzymatic loading were evaluated. Immobilized cellulases showed better hydrolysis performance when an enzyme loading of 134 mg protein/g carrier was used for immobilization at pH 9.0 for 30 minutes. Hydrolysates with a glucose content of 13.43 and 10.35 g/L were obtained when Avicel and pretreated sugarcane bagasse were used as substrate, respectively. Immobilized cellulase lost 60% of its hydrolysis performance after 8 cycles using Avicel, and 75% after 6 cycles for sugarcane bagasse. The hydrolysis performance associated with the reuse of the immobilized cellulases indicates that an improvement in the immobilization of cellulases, coupled with an improvement in the pretreatment of lignocellulosic biomass, will allow the development of a continuous hydrolysis system with the enzyme retained in the reactor.

Optimization of Carotenoids Production from Camelina sativa Meal Hydrolysate by Rhodosporidium toruloides

Several compounds on the market derive from petrochemical synthesis, and carotenoids are no exception. Nonetheless, since their applications in the food, feed and cosmetic sectors, and because of sustainability issues, carotenoids of natural origin are desirable. Carotenoids can be extracted from several plants but also from carotenogenic microorganisms, among which are yeasts. Nonetheless, to meet sustainability criteria, the substrate used for yeast cultivation has to be formulated from residual biomasses. For these reasons, we deploy the yeast, Rhodosporidium toruloides, to obtain carotenoids from Camelina sativa meal, an underrated lignocellulosic biomass. Its enzymatic hydrolysis ensures the release of the sugars, as well as of the other nutrients necessary to sustain the process. We therefore separately optimized enzymatic and biomass loadings, and calculated the yields and productivities of the obtained carotenoids. The best conditions (9% w/v biomass, 0.56% w/wbiomass enzymes) were tested in different settings, in which the fermentation was performed separately or simultaneously with hydrolysis, resulting in a similar production of carotenoids. In order to collect quantitative data under controlled chemo-physical parameters, the process was implemented in stirred-tank bioreactors, obtaining 3.6 ± 0.69 mg/L of carotenoids; despite the volumetric and geometric change, the outcomes were consistent with results from the fermentation of shake flasks. Therefore, these data pave the way to evaluate a potential future industrialization of this bioprocess, considering the opportunity to optimize the use of different amounts of biomass and enzyme loading, as well as the robustness of the process in the bioreactor.

An Electrochemical Enzyme Biosensor for Ammonium Detection in Aquaculture Using Screen-Printed Electrode Modified by Gold Nanoparticle/Polymethylene Blue

A SPEC/AuNPs/PMB modified electrode was prepared by electrodeposition and electro-polymerization. The electrochemical behavior of reduced nicotinamide adenine dinucleotide (NADH) on the surface of the modified electrode was studied by cyclic voltammetry. A certain amount of substrate and glutamate dehydrogenase (GLDH) were coated on the modified electrode to form a functional enzyme membrane. The ammonia nitrogen in the water sample could be calculated indirectly by measuring the consumption of NADH in the reaction. The results showed that the strength of electro-catalytic current signal was increased by two times; the catalytic oxidation potential was shifted to the left by 0.5 V, and the anti-interference ability of the sensor was enhanced. The optimum substrate concentration and enzyme loading were determined as 1.3 mM NADH, 28 mM α-Ketoglutarate and 2.0 U GLDH, respectively. The homemade ceramic heating plate controlled the working electrode to work at 37 °C. A pH compensation algorithm based on piecewise linear interpolation could reduce the measurement error to less than 3.29 μM. The biosensor exhibited good linearity in the range of 0~300 μM with a detection limit of 0.65 μM NH4+. Compared with standard Nessler’s method, the recoveries were 93.71~105.92%. The biosensor was found to be stable for at least 14 days when refrigerated and sealed at 4 °C.

Optimization of Carotenoids Production from Camelina Sativa Meal Hydrolysate by Rhodosporidium Toruloides

BackgroundPetrochemical synthetic dominates several markets, and carotenoids are not an exception. Since their applications in the food, feed and cosmetic sectors, carotenoids of natural origin are increasingly requested, but the production needs to be sustainable also in terms of initial feedstock. For these reasons we deployed the carotenogenic yeast Rhodosporidium toruloides to obtain such compounds from Camelina sativa meal, an underrated lignocellulosic biomass. As the process starts from hydrolyzed biomass, we separately optimized enzymatic and biomass loadings, to reduce the overall process costs. ResultsThe best conditions (9% w/v biomass, 0.56% w/wbiomass enzymes) were tested in different settings, in which fermentation was separate or co-current with the hydrolysis, showing similar carotenoids productions. The process was implemented in stirred-tank bioreactors, obtaining 3.6 ± 0.69 mg/L of carotenoids, and showing to be robust towards changes in different parameters. ConclusionsThese data pave the way to evaluate a possible industrialization of this bioprocess, considering the opportunity to optimize the use of different amounts of biomass and enzyme loading. In addition, the test in bioreactor is an additional step to further develop the proposed process.